Insulin regulates the activity and/or amount of over 40 different proteins. In some cases activity is modified by covalent modification of an existing amount of protein. In other cases insulin affects the rate of synthesis of specific proteins. Until recently little was known about this process. Fror the past four years we have been investigating how insulin decreases the rate of synthesis of phosphoenolypyruvate carboxykinase (PEPCK), the rate-limiting enzyme in gluconeogenesis. We have found that insulin selectively inhibits transcription of the PEPCk gene. This results, in sequence, in decreased nuclear mRNAPEPCK, decreased cytoplasmic mRNAPEPCK and decreased PEPCK synthesis. Three groups of studies will be used to investigate how this novel action of insulin is accomplished. These include: 1) Studies of how insulin and related molecules affect PEPCK gene transcription in cultured H4IIE hematoma cells; 2) A search for the substrance or process that couples the interaction of insulin and its receptor to this nuclear effect; and 3) An analysis of this action of insulin at the PEPCK gene level. These studies will use assays that measure the initiation and elongation of PEPCK gene transcription and will involve fusion gene analysis to search for DNA sequences involved in this effect. These studies are important because this the first example of a specific effect of insulin at the gene level. These studies should provide a prototype for other examples which are sure to follow.